Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all photosynthetic organisms, and in some heterotrophic growing bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish and birds. Colors of carotenoid range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in our diet and they play additional important role in human health. Industrial uses of carotenoids include pharmaceuticals, food supplements, animal feed additives and colorants in cosmetics to mention a few.
Because animals are unable to synthesize carotenoid de novo, they must obtain them by dietary means. Thus, manipulation of carotenoid production and composition in plants or bacteria can provide new or improved source for carotenoids.
Carotenoids come in many different forms and chemical structures. Most naturally occurring carotenoids are hydrophobic tetraterpenoids containing a C40 methyl-branched hydrocarbon backbone derived from successive condensation of eithght C5 isoprene units (IPP). In addition, rare carotenoids with longer or shorter backbones occur in some species of nonphotosynthetic bacteria. The term “carotenoid” actually include both carotenes and xanthophylls. A “carotene” refers to a hydrocarbon carotenoid. Carotene derivatives that contain one or more oxygen atoms, in the form of hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functional groups, or within glycosides, glycoside esters, or sulfates, are collectively known as “xanthophylls”. Carotenoids are furthermore described as being acyclic, monocyclic, or bicyclic depending on whether the ends of the hydrocarbon backbones have been cyclized to yield aliphatic or cyclic ring structures (G. Armstrong, (1999) In Comprehensive Natural Products Chemistry, Elsevier Press, volume 2, pp 321–352).
Carotenoid biosynthesis starts with the isoprenoid pathway and the generation of a C5 isoprene unit, isopentenyl pyrophosphate (IPP). IPP is condensed with its isomer dimethylallyl pyrophophate (DMAPP) to form the C10, geranyl pyrophosphate (GPP), and elongated to the C15, farnesyl pyrophosphate (FPP). FPP synthesis is common to both carotenogenic and non-carotenogenic bacteria. Enzymes in subsequent carotenoid pathways generate carotenoid pigments from the FPP precursor and can be divided into two categories: carotene backbone synthesis enzymes and subsequent modification enzymes. The backbone synthesis enzymes include geranyl geranyl pyrophosphate synthase, phytoene synthase, phytoene dehydrogenase and lycopene cyclase, etc. The modification enzymes include ketolases, hydroxylases, dehydratases, glycosylases, etc.
Carotenoid ketolases are a class of enzymes that introduce keto groups to the ionone ring of the cyclic carotenoids such as β-carotene to produce ketocarotenoids. Ketocarotenoids include astaxanthin, canthaxanthin, adonixanthin, adonirubin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, 4-keto-gamma-carotene, 4-keto-rubixanthin, 4-keto-torulene, 3-hydroxy-4-keto-torulene, deoxyflexixanthin, myxobactone. Astaxanthin was reported to boost immune functions in humans, and, reduce carcinogenesis in animals. Unlike genes in the upstream isoprenoid pathway that are common in all organisms, the downstream carotenoid modifying enzymes are rare. Two classes of ketolase, CrtW and CrtO, have been reported. The CrtW is a symmetrically acting enzyme that adds keto-groups to both rings of β-carotene (Hannibal et al., J. Bacteriol. (2000) 182: 3850–3853). Fernández-González et al. (J. of Biol. Chem. (1997) 272;9728–9733) has discovered another ketolase enzyme, CrtO, from Synechocystis sp. PCC6803 that adds a keto-group asymmetrically to only one β-carotene rings. The crtO gene from Haematococcus pluvialis has been transferred to tobacco pant to express astaxanthin in the plant (Mann et al., (2000) Nature Biotechnology, 18:888–892).
Although the genes involved in carotenoid biosynthesis pathways are known in some organisms, genes involved in carotenoid biosynthesis in Rhodococcus bacteria are not described in the existing literature. However, there are many pigmented Rhodococcus bacteria suggesting that the ability to produce carotenoid pigments is widespread in these bacteria. Carotenoids of Rhodococcus have been structurally characterized in Rhodococcus as described by Ichiyama et al., (Microbiol. Immunol. (1989), 33:503–508).
The problem to be solved therefore is to isolate sequences involved in carotenoid biosynthesis in Rhodococcus for their eventual use in carotenoid production. Applicants have solved the stated problem by isolating a gene, crtO, from a Rhodococcus erythropolis AN12 strain containing an open reading frame (ORF) encoding a ketolase enzyme that contains 6 conserved diagnostic amino acid motifs that are the characteristic of this type of ketolase enzymes.